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An ALYREF-MYCN coactivator complex drives neuroblastoma tumorigenesis through effects on USP3 and MYCN stability.

ABSTRACT: To achieve the very high oncoprotein levels required to drive the malignant state cancer cells utilise the ubiquitin proteasome system to upregulate transcription factor levels. Here our analyses identify ALYREF, expressed from the most common genetic copy number variation in neuroblastoma, chromosome 17q21-ter gain as a key regulator of MYCN protein turnover. We show strong co-operativity between ALYREF and MYCN from transgenic models of neuroblastoma in vitro and in vivo. The two proteins form a nuclear coactivator complex which stimulates transcription of the ubiquitin specific peptidase 3, USP3. We show that increased USP3 levels reduce K-48- and K-63-linked ubiquitination of MYCN, thus driving up MYCN protein stability. In the MYCN-ALYREF-USP3 signal, ALYREF is required for MYCN effects on the malignant phenotype and that of USP3 on MYCN stability. This data defines a MYCN oncoprotein dependency state which provides a rationale for future pharmacological studies.

SUBMITTER: Nagy Z

PROVIDER: S-EPMC7994381 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWhile RNA-binding proteins (RBPs) are known to affect RNA homeostasis during cancer cell initiation and development, their characteristics and biological function in glioblastoma (GBM) remain unclear.MethodsDifferences in RBP expression were explored by differential analysis of The Cancer Genome Atlas-GBM and Genotype-Tissue Expression (GTEx) datasets. Real-time PCR was conducted to verify the expressional levels of Aly/REF export factor (ALYREF) in normal brain and GBM tissues. Proliferative assays were performed to investigate molecular functions of ALYREF in GBM cells in vitro and in vivo. Real-time PCR and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the ALYREF downstream signaling pathways. A chromatin immunoprecipitation (ChIP) assay was performed to identify key transcriptional factors that regulate ALYREF expression at RNA level. UV crosslinking, immunoprecipitation (CLIP) and RNA stability assays were conducted to reveal the bound RNAs and their stability regulated by ALYREF.ResultsThe results showed that ALYREF is frequently increased in GBM tissues, and its mRNA expression is regulated by the MYC proto-oncogene, bHLH transcription factor (MYC). Inhibition of ALYREF expression decreased GBM cell proliferative ability in vitro and tumor formation in vivo. KEGG analysis revealed that high ALYREF expression in GBM tissues was enriched in the upregulation of oncogenic pathways such as the Wnt/β-catenin signaling pathway. The CLIP assay showed that ALYREF drives GBM carcinogenesis by binding to and stabilizing MYC mRNAs. Overexpression of MYC restored the oncogenic property of ALYREF-deficient GBM cells.ConclusionOur data showed that ALYREF is regulated by MYC at the transcriptional level. ALYREF drives GBM cell proliferation by activating the Wnt/β-catenin signaling pathway and stabilizing MYC mRNA, suggesting that an ALYREF-MYC positive feedback loop might be a potential therapeutic target for treating GBM patients.

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An ALYREF-MYCN coactivator complex drives neuroblastoma tumorigenesis through effects on USP3 and MYCN stability.

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